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• MATERIAL AND METHODS
• Isolation of Bacteria
• PCR Primer design for phage-related integrase and transposase gene
• Detection of bacteria using specific primers
• Validation of Xoo by specific duplex primers
• Detection of bacteria in rice field water and in plants
• RESULTS
• Primer design and specificity
• Validation of PCR specific primers in collected Xoo strains
• Detection of Xoo in rice leaves and rice field water
• Discussion

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight (BB) in rice. Bacterial cells on leaf surfaces enters the rice leaf by either swimming passively through the fluid oozing from hydathodes in the morning and spreading systemically in the plant through the xylem, or it enters directly into the xylem through wounds (Nino-Lui et al., 2006). BB of rice has become an important problem affecting rice production worldwide. Some anti-bacterial agents (bactericides) have been developed and used to control the disease but none is highly effective and economical. Breeding rice varieties with genetic resistance for BB was found to be the most effective, economical, and environmentally benign method for the control of BB (Mew, 1987). However, the breeding programs might cause the evolution of new pathogenic races leading to outbreak of this disease. (Browing and Frey, 1969). Several approached including biochemical tests (Vera Cruz et al., 1984), serological assays (Benedict et al., 1989), fatty acids, and metabolic profiling (Chase et al., 1992; Jones et al., 1993) have been used to identify the pathogen. However, these assays have limitations such as lack of sensitivity and specificity. Due to problems encountered in conventional methods, polymerase chain reaction (PCR) technique has found a wide application in the detection of plant pathogenic bacteria (DeParasis and Roth, 1990; Blakemore et al., 1992; Goodwin and Nassuth, 1993; Lopes and Damann, 1994; Prosen et al., 1993; Schierwater and Ender, 1993; Seal et al., 1993). PCR technique has provided a highly sensitive method, where a few nanograms of template DNA are enough to obtain simple and reproducible amplification patterns from complex genomic DNA (Welsh and MeClelland, 1990). In this study, specific primers of Xoo were designed from phage-related integrase and transposase gene region of Xoo. In addition, the primer set showed high sensitivity and specificity for PCR-based detection of the pathogen in paddy field water and plants of rice.

MATERIAL AND METHODS

Isolation of Bacteria

Bacteria isolated from infected rice leaves by following the method of Adhikari et al., (1995). Bacteria that have yellow circular colonies with entire margins and smooth waxy and shiny surface were isolated. Single bacterial colonies of each strain were isolated. The isolated colonies were further purified on peptone sucrose agar (PSA) medium (Ou, 1985). Afterwards bacterial cells were preserved in 15% glycerol at −70˚C and PSA slants at 4˚C as a source for further work. For long-term storage, the isolate were preserved in the silica gel at 4˚C.

PCR Primer design for phage-related integrase and transposase gene

Xoo PCR Primers were designed by aligning the 401 and 492 bp variable region of phage-related integrase and transposase A gene of XOO. The sequence of phage-related integrase(AE013598.1 REGION: 637152..638426) and transposase A (AE013598.1 REGION: 46077..47102) gene of XOO downloaded from NCBI and 10 primers designed for each gene with the NCBI pick software. Out of 10 primers only one primer of phage-related integrase and one primer of transposase A gene was able to detected the Xoo (Table 1). A primer set showing specific amplified banding pattern of Xoo analysis was chosen.

Table 1. The sequence of Xoo primers.

Table 2. The PCR condition for Xoo detection.

Table 3. The result of specific test of duplex primer for Xoo detection.

Detection of bacteria using specific primers

Each PCR reactions contained 1 μl of DNA template, 0.4 μl of 80mM dNTP, 0.6 μl of 150m M MgCl2, 2 μl of 10 X Taq polymerase buffer 1 μl of XOP-F (5-CGGTCTGCTCAATGAGGAAGA- 3) and XOP-R2 (5- TGCAATTGGTGTTCTCCAGG- 3) and XOT-F (5- GTCATAGGTGAGGCTTCCC -3) and XOT-R2 (5- AGTGCGATCTTTCAGCAGG -3) (Table 1) each primer (4pmols each) and 0.3μl (2 units) Taq DNA polymerase (TNT Research, Inc. Korea). PCR with specific primers XOP-F, XOP-R, XOT-F and XOT-R was performed (Applied Biosystems, 2720 Thermo cycler; USA). Samples were amplified through 35 cycles, with an initial denaturation at 94˚C for 4 min, followed by 15 sec at 94˚C, 15 sec of annealing at 63˚C for upgrade specificity and get rid of miner fragments, 30 sec of extension at 72˚C and final extension at 72˚C for 5min. Nine microliters of each amplified product was fractionated on a 1% agarose gel in 1 X TAE buffer. Gel was stained with ethidium bromide and photographed under UV light.

Validation of Xoo by specific duplex primers

We have collection of 218 Xoo strains during 2001-2010 (Table 4). We validated these dual PCR-specific primers for the validation of 218 Xoo strains. We used the same PCR reaction as for the detection of Xoo strain.

Table 4. Strain of the Xoo collected during the 2001-2010 used for the validation of duplex PCR primer.

Detection of bacteria in rice field water and in plants

The infected leaf with Xoo strains cut into 1 × 1 cm and then these leaves were vigorously shaken (160 rpm) for about 30min in 20ml of water. A 2 μl portion of each sample was used for PCR in the same manner as described as above. 

RESULTS

Primer design and specificity

On the basis of these nucleotide sequences, dual PCR primers XOP-F, XOP-R, XOT-F, and XOT-F (described in Materials and Methods) was synthesized for specific amplification of the phage-related integrase and transposase A gene in X. oryzae pv. oryzae. Using these primers, two 401 bp and 492 bp fragments were amplified from lysed cells of all strains of X. oryzae pv. oryzae examined in this study (Fig. 1). No amplification was obtained from other than Xoo, however 492bp fragment was amplified from P. savastanoi pv. phaeolicola (Fig. 2). The detection sensitivity limit of PCR for X. oryzae pv. oryzae using these primers was 2^-6 ng DNA concentration (Fig. 3).

Fig. 1. Agrose gel electrophoresis of PCR products from X. oryzae. pv. oryzae and distilled water (DW) using duplex primers XOP-F, XOP-R, XOT-F, and XOT-F. M; Maker, 1; HB01009, , 2; HB01013 3; KACC01331, 4; HB01014, HB01015, 5; HB02010, 6; HB03011, 7-8; DW.

Fig. 2. Agrose gel electrophoresis of PCR products from X.oryzae. pv. oryzae and other species bacterial strains using duplex primers XOP-F, XOP-R, XOT-F, and XOT-F.M; Maker, 1-2; Xoo DNA, 3; DW, 4; Escherichia coli, 5; Agrobacterim, 6; Pectobacterium caratovora subsp. cartovorum, 7; P. atrosepticum, 8; p. putida, 9; P. syringae, 10; P. savastanoi pv. phaeolicola, 11; P. savastanoi pv. savastanoi, 12; P. marginalis pv. Marginalis.

Fig. 3. Amplification of PCR product at different DNA concentration (in ng) of the isolated Xoo.

Validation of PCR specific primers in collected Xoo strains

We identified the two bends of 401 bp and 492 bp size in all the Xoo strains collected during the 2001-2010 year. The PCR product of some selected Xoo strains shown in the Fig. 4.

Fig. 4. Validation of some selected Xoo strain by PCR-specific primers collected during the year of 2001-2010. Strain name HB06336, HB06337, HB06340, HB06344, HB06348, HB06356, HB06376, HB06384, HB06388, HB06389, HB06411, HB06416, HB06442, HB06472, HB07015, HB07021, HB07023, HB07029, HB07033, HB07036, HB07040, HB07046, HB07062, HB07086.

Detection of Xoo in rice leaves and rice field water

PCR products were also obtained from rice leaves infected with X. oryzae pv. oryzae using dual PCR-specific primers XOP-F, XOP-R, XOT-F, and XOT-F. Using these primers, two 401bp and 492bp fragments were successfully amplified from infected rice leaves and rice paddy field water. On the contrary, no product was observed in the case of non-infected plants. 

Discussion

Japonica rice cultivars exhibit high susceptibility to BB disease due to their narrow genetic diversity. A new BB race K3a caused significant yield loss in Korea in 2003 (Noh et al. 2003). It is interesting to know how the Xoo pathogen behaves in the absence of host plants, and more effective method to assay the pathogen is needed. A phage specific detection for Xoo has been previously reported which shows high specificity and sensitivity (Yoshimura, 1963) (10²cfu/ml). However, these include the complicate and time-consuming steps for analyzing many samples. In contrast, PCRbased detection of Xoo targeting the phage-related integrase and transposase A gene is cost effective and time saving. Development of specific DNA probes and primers for detection has been reported for a number of plant pathogenic bacteria (Hartung, 1992; Rasmussen and Reeves, 1992). Some of these DNA probes and primers were developed from random screening of cloned fragments or insertion sequences of total genomic DNA (Gilbertson et al., 1989; Johansen et al., 1989; Thompson et al., 1989). This study focused on the phage-related integrase and transposase A gene for the detection and identification of X. oryzae pv. oryzae with the use of PCR assay. Two 401bp and 492bp fragments were found in all strains isolated from diverse regions in Korea following amplification by PCR using dual specific primers XOP-F, XOP-R, XOT-F, and XOT-F. To identify Xoo it is required to amplify two PCR fragments using this primer set, however, P. savastanoi pv. phaeolicola was amplified at 492bp in this study, so that it is appeared that there is no problem when we use the primer to identify the Xoo. In this report, we developed the PCR-based assay and specific markers for detection of the pathogen. This method has several notable advantages. It takes only 3 hr to detect the pathogen in plant tissues and the sensitivity is sufficiently high, similar to that of methods previously reported (Li and De Boer, 1995; Maes et al., 1996; Miyoshi et al., 1998; Takeuchi et al. 1997). Therefore, the PCR technique described here using dual specific primers XOP-F, XOP-R, XOT-F, and XOT-F should serve as an effective tool for detection of Xoo in the field.